Transgenic mice are vital tools in both basic and applied research. Virtually all techniques used to generate transgenic and knock-out mice relay on Embryo Transfer (ET) as a means of producing animals from embryos manipulated in vitro. Strain rederivation by ET is also the most frequent method of eliminating disease pathogens and parasites from laboratory strains of mice. Presently, the ET procedure requires the preparation of pseudopregnant females by mating them with vasectomised males. Only pseudopregnant foster mothers can subsequently implant graft embryos and until now, mating of females with vasectomised males is the only reliable way of inducing pseudopregnancy in mice. The vasectomy procedure is associated with moderate to high pain and must be carried out by trained personnel with the animal receiving full anaesthesia followed by a recovery period and analgesic regime. Eliminating of the need for vasectomy would not only simplify the ET process but is also highly desirable from the animal welfare point of view. One possible strategy would be to use male mice that are capable of copulation but are unable to produce offspring due to a genetic defect in sperm maturation.
To date, a number of genes have been identified which when mutated cause male sterility. Most of these genes are involved in control of DNA recombination, DNA repair or differentiation of the male reproductive tract or the male germline (Bellve A R. The Molecular Biology of Mammalian Spermatogenesis, ed. Finn C A., Oxford Univ. Press, London, Vol. 1, pp. 159-261; Brugmans L et al., Mutat. Res., 2007, 614(1-2):95-108; Jaroudi S & SenGupta S., Mutat Res., 2007, 635(1):53-77). Unfortunately, most of these strains are also associated with decreased viability, susceptibility to cancer or other diseases. In addition, strains harbouring such mutations are not good candidates for vasectomy replacement since these recessive traits require breeding and reliable genotyping of heterozygous (fertile) versus homozygous (sterile) male mutant mice. Classical dominant mutations causing male sterility have been described but also none of them appear to be ideally suited for the purpose of replacing surgically vasectomised males in a laboratory environment. These mutants include the Dominant spermiogenesis defect (Dspd) which results from a reciprocical translocation between chromosomes 14 and 7 (Kai M. et al., Biol Reprod., 2004, 70(4):1213-21) as well as two randomly generated insertional mutations named Lacking vigorous sperm (Lvs) (Magram J. & Bishop J M., Proc Natl Acad Sci USA., 1991, 88(22):10327-31) and Dominant male sterility (Dms) (Meng X, et al., Biol Reprod., 2002, 66(3):726-34), respectively. Since the genes causing the sterility have not been identified, it is impossible to reproduce the phenotype in other strains. Furthermore, the lack of a phenotypic marker associated with the sterility trait adds the burden of identification of transgenic offspring by genotyping methods.
Dominant male-sterility has also been described in mice overexpressing transgenes. For example, ubiquitous overexpression of the Retinoic acid receptor alpha (Rarα) causes dominant male-sterility due to severe malformation of the epithelium of the reproductive tract (Costa S L, et al., Biol Reprod., 1997, 56(4):985-90). Unfortunately, the perturbation of the epithelium also prevents the formation of the ejaculate. Hence, sterile males fail to produce a copulatory plug, also known as mating plug, copulation plug, vaginal plug, or sphragis, upon mating making the identification of pseudopregnant females virtually impossible.
Overexpression of the Retinits pigmentosa GTPase regulator (Rpgr) protein also leads to male infertility in mice due to defects in flagellar assembly (Brunner S., et al., Biol. Reprod., 2008, 79(4):608-17). Finally, it was shown that premature overexpression of mouse Protamine-1 (Prm1) can cause dominant male sterility. The nuclear protein Prm1, together with its homolog Prm2, play crucial roles in the nuclear reorganization of elongating spermatids during the final stages of sperm development. Protamine proteins ultimately replace the bulk of histones from the genomic DNA and mediate extreme condensation of the sperm genome (Peschon J J. et al., Proc Natl Acad Sci USA., 1987, 84(15):5316-9). The Prm1 gene is transcribed in round spermatocytes, while the resulting mRNA is translated about 4 days later in late elongating spermatids. This translational delay is brought about by regulatory elements residing in the 3′UTR of the Prm1 mRNA (Zhong J., et al., Biol. Reprod., 2001, 64(6):1784-9). Previous studies have shown that premature translation of Prm1 in round spermatids, brought about by replacement of the Prm1 3′UTR with a heterologous 3′UTR, leads to defects in spermatid maturation and results in the absence of sperm and subsequent male sterility (Lee K. et al., Proc. Natl. Acad. Sci. USA., 1995, 92(26):12451-5).
At the time of the invention, there was a need in the art for genetic alternatives for e.g. surgical vasectomy. Some solutions had been proposed. However, some problems still remained. First of all, the sterility phenotype was not fully penetrant in all lines tested, and second, the absence of a visible marker made the identification of mice carrying the transgene difficult.
The present inventors developed a mouse strain that accommodates all these requirements. Transgenic expression of a fusion protein induces dominant sterility with full penetrance. Moreover, the fact that one of the partners of the fusion protein can be a label greatly facilitates the identification of the mice carrying the fusion protein. Alternatively, genetically linking the expression of the fusion protein to ubiquitous label expression in the soma, also provides a reliable and cost-effective procedure, outperforming e.g. the classical surgical vasectomy methodology.